A variety of separative, synthetic, and enzymatic or otherwise catalytic processes use beds of particulate material with transport of reactants, reagents and products or eluants in solution through the bed. In addition, many reactions are known in which the products are separated by concentration in one of two or more phases. These processes include, among others, ion exchange chromatography, gel filtration, ion exclusion chromatography, affinity chromatography, separations based on hydrophobicity, purification based on hybridization, peptide synthesis, oligonucleotide synthesis, and polysaccharide synthesis including combinations of the last three. These processes may be carried out on a small scale for analytical purposes or process design, and are then often scaled up for preparative work. In nearly all examples the solid phase particulates are packed in a closed column with a porous frit on the lower end, an optional frit at the top, and with fluid-connections at both ends so that liquid can flow in either direction through the bed. To achieve efficiency and high resolution with solid phase supports, all volume elements of all fluids should flow through paths of identical composition and nearly identical length, and all particles in the bed should be exposed to the same succession of liquids under the same conditions.
In all instances involving solid phase systems, some interaction occurs between the solutes run through the bed and the particles composing the bed. This interaction may be based on secondary forces (ionic, hydrophobic, or on immunochemical interactions, or base pairing) or primary valencies as when amino acids or nucleotides are added to a growing chain on the solid phase support, or when immobilized enzymes cleave substrates flowing through the bed, or when enzymes in solution react with substrates attached to the packing. In addition, solvents or reagents of successively differing composition which dissociate adsorbed or otherwise attached bound molecular species, or which cleave off protective groups, or compounds including polymers which have been synthesized on the support may be made to flow through the support. The dissociated or cleaved substances then are free to flow out of the bed in flowing liquid.
It is a common experience that when processes using particulate beds are scaled up for any purpose, resolution and efficiency are lost. At both bench level and preparative level the reactions occurring in and around each particle or element of membrane support should, ideally, be the same. The differences seen during scaling up are primarily in the uniformity of flow through the bed, in the length of the fluid path through the bed, in the length of time a solid phase bead is exposed to a given reagent, and in the volume of spaces above and below the columns where fluid is funnelled into the attached lines, but where, in conventional systems, mixing and loss of resolution may occur. Some of the differences are due to differences in the rate of flow in different small volume elements of the bed termed microanomalous flow, and band tilting and mixing in end spaces, termed macroanomalous flow.
In addition to microanomalous flow, which is largely due to differences in the size and shape of individual bed particles and in local packing density and geometry, density differences and density inversions between sequentially employed solutions may also prevent ideal flow since ordinarily no attention is paid to liquid density differences. As previously demonstrated in the centrifugal fast chromatograph (U.S. Pat. Nos. 4,900,435 and 4,900,446), careful and rational control of density and use of density gradients will control both macro- and micro-anomalous flow if separations are carried out in a centrifugal field. The basic principles of centrifugal stabilization of density gradients, and of reversal of flow to regenerate columns, also in a centrifugal field, have therefore been previously described. The centrifugal fast chromatograph is an analytical device in which a number of columns are run in parallel using very small samples.
In chromatographic separations, more capacity is needed at the end of the column where the sample is applied, and the thickness of the initial sample zone is partially dependent on column capacity. As successive peaks are eluted, and as they move down the column, less column capacity per unit length is required. Thus, for chromatography, advantages accrue if the column is in the form of a sector, with the sample applied to the large end, and the effluent withdrawn at the narrow end. In a zonal centrifuge (National Cancer Inst. Monograph No 21, 1966) flow is arranged to be radial, and may be from the edge of a sector-shaped compartment toward the center. The desired flow configuration can be achieved by the present invention.
The requirements for peptide or oligonucleotide syntheses are quite stringent. Antisense oligonucleotides, which are complimentary to RNA or DNA strands of cells, hold the promise of controlling specifically the expression of individual genes, and therefore are of interest as anti-viral agents against HIV and other pathogens, for controlling and even reversing genetic diseases, and for treating cancer--all by translational or hybridization arrest; and by serving as carriers for active groups. To achieve specificity in intracellular hybridization, oligonucleotides approximately 15-18 or more nucleotides long are required. Since native or natural oligonucleotides are rapidly degraded in a biological environment, a variety of modified oligonucleotides have been proposed (Bioconjugate Chemistry 2:165 (19990)). Kologram quantities of highly purified and sequence-specific oligonucleotides (so-called oligos) will be required for large scale animal and clinical trials, and ultimately for clinical use. Oligos are now synthesized in milligram to gram scales with existing bench top equipment. At present, approximately 10 grams of solid support such as controlled pore glass is required for the synthesis of 1 gram of crude material. To synthesize 1 kilo in a single operation would, therefore, require 10 kilos of support, at a cost estimated variously at between $300,000 and $1,000,000 dollars. Clearly, methods for reducing cost and increasing yields are of interest for the synthesis of not only oligonucleotides but also peptides and polysaccharides. Cost reductions and yield enhancements are also desired for other preparative and separative processes.
The most important consideration in oligonucleotide synthesis is yield of pure product, which is dependent on the efficiency of the coupling reaction, the absence of failure sequences, and on minimizing side and degradative reactions. Hence great effort has been expended on the development of efficient chemical procedures and reagents, and on optimizing the time required for each step in the synthesis cycle. The effect of overall efficiency is illustrated by calculating the overall yields for different effective coupling cycle efficiencies. If the average cycle efficiency is 99%, the yield after 20 cycles (which would yield an oligo 21 nucleotides long, since the first or "seed" nucleotide is already attached to the solid support at the outset) would be 83% of the theoretical maximum one. For cycle efficiencies of 98%, and 95%, the yields would fall to 67%, and 36%. Clearly every factor affecting yield is important.
Oligonucleotide synthesis typically involves a series of eleven steps (including washes), the first of which is deprotection of the seed nucleotide (generally removal of a dimethoxytrityl group which protects a terminal reactive group on the deoxysugar of a nucleotide). This is done in acid, and the acid and cleaved trityl group are removed by three washes which also involve a change of solvent from dichloromethane to dry acetonitrile. Nucleotide addition is then done using an activated nucleotide such as a phosphite triester, for example, a deoxynucleoside 3'-phosphoramidite in the presence of an activator such as tetrazole. The addition reaction is very rapid and essentially complete in five minutes (Oligonucleotides, J. Cohen, ed., CRC Press, 1989, pp 7-24). After a further wash, those reactive nucleotides remaining (i.e., those to which no nucleotide was added in the previous coupling step) are capped with acetic anhydride. Following an additional wash, an aqueous oxidizing solution is added to oxidize the phosphorous of the added nucleotide, and the support is again washed with a change of solvent from acetonitrile to dichloromethane. This cycle of solutions is repeated for each addition. For the synthesis of an oligo 21 nucleotides long (a so-called 21 mer), 221 or more discrete solutions flow through the solid phase reaction bed.
Several of these solutions are incompatible. Thus, exclusion of water is essential in the coupling step, but the oxidation solution is 20% water by volume. The deprotection solution removes trityl groups, but deprotection must be prevented during the coupling step when the presence of a trityl group on the added nucleotide is essential. The iodine from the oxidizing step must also not be present during coupling, and the capping reagents must be absent between deprotection and coupling. Hence there is extensive washing between the reactive reagents. All of the reagents are expensive, and those remaining after synthesis must be suitably disposed of, also at considerable expense. Any advance which will reduce the volume of reagents required without decreasing yield is therefore very desirable. Recently (Japanese Patent No. 6,379,895) the efficient synthesis of a 90 nucleotide long oligomer (a 90 mer) has been demonstrated without washing between steps. This appears to be due to efficient exchange of one solution with another with minimum reagent trailing, and suggests that if the flow of solutions through a solid support bed could be very precisely controlled and trailing of one solution into the next minimized, that wash volumes could be either diminished or eliminated.
The reaction times involved in specific synthetic steps also create problems during scale up of oligonucleotide synthesis. Deprotection is usually done in 3 minutes, coupling (nucleotide addition) in 5, capping in 2, and oxidation in 1, with washes lasting either 0.5 or 1 minute. If a bed volume is scaled up to 1 liter, for example, it will be difficult to achieve flow rates which will allow such rapid solution changes. Further, reagents diffuse into and out of the pores and interstices of solid phase particles at rates which depend on both the particle and pore sizes, the temperature, the molecular weights of the solutes, and the viscosity of the solvent and are never instantaneous. When one solvent succeeds another in a porous or adsorbent bed, there will therefore be some trailing of adsorbed or included solutes from the previous solution. Hence means for controlling flow, for preventing non-ideal flow, and for keeping interfaces between succeeding fluids as sharp as possible are essential. If the very same schedules used on a bench scale are to be applied to a very large system, then very fast flow rates and large volumes of solution would be required.
The reason for the time limitations on some of the steps is either that side reactions accompany excess dwell time, or activated ingredients become exhausted. An objective in scale up, therefore is to provide sufficient reaction time to carry a reaction essentially to completion, but insufficient time for deleterious reactions to occur.
Similar requirements and limitations occur in the solid phase synthesis of peptides. With other uses of packed beds, scale up involves loss of resolution for reasons mentioned, and usually some dilution of the product.
Some stages of a sequential series of steps in a separation or synthesis are more time dependent than others. Chromatographic separations are generally dependent on the rate of diffusion of the sample components into and out of the chromatographic beads, affinity separations on the rate of diffusion of the substance being purified and the binding energy between the ligands in solution and the adsorbing surfaces, while synthetic procedures depend on the rate of synthetic reactions. However the efficiency of each of these processes are improved if both microanomalous and macroanomalous flow are prevented. Other steps, such as pH changes during regeneration, temperature change, or solvent changes between steps can also accomplished much more rapidly and efficiently if flow is optimized. Further, it is advantageous to be able to change the flow rates markedly during a procedure without producing disturbances in flow. In addition, excess reagent is required in many systems where many and long fluid lines are required to connect and interconnect complex valve systems.
In conventional procedures using particulate beds, careful attention must also be given to removing gas bubbles which may already exist in the packing, and in preventing their formation from dissolved gasses. In some instances, degassing of solutions is required.
Scale up of biosynthetic and bioseparative processes therefore involves problems of scale which reduce yields, and degrade separations. These and other problems are addressed by the centrifugal processor of the present invention.